Ca2+ has both positive and negative effects on T cell signalling. On the positive side, Ca2+ activates diverse enzymes necessary for productive activation of T cells. On the negative side, Ca2+ has been implicated in feedback attenuation of signalling pathways and in the development of T cell anergy. Ca2+ also influences cytoskeletal function in multiple ways, both positive and negative. The long-term objective of this proposal is to understand the crosstalk among Ca2+, the immunological synapse, and the cytoskeletal changes that accompany T cell signalling. Aim 1 will study the role of the cytoskeleton in Ca2+ signalling and immunological synapse formation in T cells during a physiological response to antigen/MHC. A) TCR-transgenic and gene-disrupted T cells (e.g., Vav-/-, WASP-/-), available from colleagues in the programme, will be used to ask how Vav, WASP, WIP and other molecules in the actin polymerization cascade influence the early and late stages of Ca2+ mobilization and activation of the transcription factors NFAT, NFkappaB and AP-1 (Fos-Jun). The integrity of signalling pathways leading to PIP2 generation, IP3 generation and PLC gamma activation will be evaluated. B) Formation and stabilization of the immunological synapse will be examined in the mutant T cells. Aim 2 will examine the negative effects of Ca2+ on T cell function. It is known that sustained Ca2+ mobilization is accompanied by the development of an anergic state, in which T cells become unresponsive to further stimulation with an antigen. As recently shown in our laboratory, these conditions result in activation of specific proteases and E3 ligases and lead to targeted destruction of specific signalling molecules downstream of the TCR. Moreover, unidentified signalling mechanisms are required in addition to sustained Ca2+ influx to activate proteolysis. These studies will be extended. A) The signalling mechanisms that synergise with calcium mobilization to cause proteolytic activation in anergic T cells will be identified. B) The range of signalling proteins that are targets for proteolytic degradation in anergy will be identified, with a specific focus on proteins in the CD28/ PKCtheta/ Vav/ WASP/ WIP/ actin polymerization pathway. C) Formation and stability of the immunological synapse in anergic T cells will be examined using the techniques described in Aim 1. D) The roles of C2-domain-containing E3 ligases in anergy-associated proteolysis will be explored. E) The role of proteolysis in T cell tolerance in vivo will be explored.